Summary: It is clear that B cells play important Ab-independent roles either promoting or inhibiting immune responses through the opposing activity of regulatory B cells (Bregs) and proinflammatory effector B cells (Beff). This is likely to explain observations that B cell depletion with anti-CD20 can rapidly improve auto- immune diseases, such as RA and MS, without depleting auto-Ab. Yet in other patients, disease is worsened. Moreover, peri-transplant depletion of B cells can markedly increase acute rejection in renal allograft recipients and chronic vasculopathy in heart transplantation. These contradictory results are likely due to the presence of both Bregs and Beff, and not knowing which predominates at a given time, in a given clinical setting, or in a given patient. A similar dichotomy is present in mice, where B cell depletion/deficiency can either inhibit or promote autoimmunity and allograft rejection. We contend that targeting B cells in autoimmune and transplant patients would have far better results if Beff were selectively targeted and Bregs were left intact. Unfortunately, little is known about Bregs, and even less is known about Beff cells. In mice, B cells expressing pro- inflammatory cytokines such as IL-6 and IFN? play a key role promoting autoimmune responses in EAE and proteoglycan-induced arthritis. Moreover, in response to various infections, B cells exhibit rapid and transient innate-like protective responses through expression of TNF?, IFN?, IL-2, and IL-17. However, it is unknown whether, or how, any of these responses relate to one another, because no phenotype for such Beff has been established and individual cytokines were examined in isolation. Thus, major aspects of Beff biology, including what regulates their differentiation and cytokine expression, are completely unknown. We have now discovered that TIM-4 identifies Beff that express inflammatory cytokines including IFN? and IL-17, and accelerate allograft rejection. Eliminating expression of these cytokines by B cells, reverses this proinflammatory role, but can also markedly inhibit the alloimmune response and promote tolerance. Moreover, some cytokines affect the expression of others. Notably, IL-17 is not only a potent effector cytokine, but is essential for Beff to develop an inflammatory rather than regulatory program. Finally, TIM-4 ligation inhibits expression of proinflammatory cytokines. Understanding the role of TIM-4+ Beff in priming the immune response and identifying how this response can be regulated to promote tolerance, are key and unique aspects of this proposal. These results are likely to provide a unifying framework for understanding Beff cells, and will have major impact on the field. In Aim 1 we will determine whether TIM-4+ Beff express a ?proinflammatory module? comprised of additional effector molecules and transcriptional regulatory elements, and determine key aspects of its regulation. In Aim 2 we will define key mechanisms by which TIM-4+ Beff promote allograft rejection. In Aim 3 we will determine how TIM-4 signaling regulates expression of the Beff pro-inflammatory module. This work will greatly enhance our understanding of Beff biology and provide therapeutic insights highly relevant to immune tolerance.